The endoplasmic reticulum (ER) is the eukaryotic organelle responsible for the production and processing of proteins destined for either secretion or membrane localization. Any deficiency in ER protein processing triggers adaptive cellular signaling for repair of the ER production pathway. This adaptation is mediated through changes in gene expression and cell physiology. A severe or sustained defect in protein processing will ultimately induce ER-initiated signals that activate apoptosis. Humans afflicted with abnormalities in the processing of proteins in the ER suffer from a broad array of devastating cellular abnormalities and disease (diabetes, cystic fibrosis, birth defects, neurodegenerative disease). In this study, cultured mammalian cells will be used to examine ER stress-induced signaling and apoptosis. The characterization of ER stress and its signaling mechanisms will offer vital insight into the cell biology of stressed cells, and will be valuable to our understanding of diseases that result from abnormal ER function Glycosylation is the covalent attachment of sugars to nascent proteins and is vital for the normal folding and function of ER synthesized proteins. Deficient glycosylation activity in the ER results in an accumulation of misfolded proteins, inducing an ER stress response. This proposal requests funding to extend our previous NIH-funded work which characterized ER stress-induced apoptosis in cells experiencing a defect in protein glycosylation. This research will test the hypothesis that a loss of protein glycosylation activity generates an apoptotic signal via release of ER Ca2+ to the cytosol. Preliminary data indicate that this Ca2+ release is mediated by the activity of the inositol 1,4,5-trisphosphate receptor in the ER membrane. To examine these preliminary finding more carefully, defects in ER function will be induced using chemical toxins and RNA interference. Additionally, we propose to examine the cellular events downstream of this ER stress signal, using a proteomics-based approach to assess the ER stress response. PUBLIC HEALTH RELEVANCE This project examines cellular signaling pathways that are initiated by stress within the endoplasmic reticulum (ER), the organelle responsible for protein biosynthesis. Deficiencies in ER function can induce signaling that has a profound affect upon cell survival and function, and have been linked to severe human disease. The characterization of ER stress and its signaling mechanisms will offer vital insight into the cell biology of stressed cells, and will be valuable to our understanding of diseases that result from abnormal ER function [unreadable] [unreadable] [unreadable]